*Target (protein/gene name): serine/threonine protein phosphatase 2b (catalytic subunit), putative (Plasmodium vivax) *NCBI Gene # or RefSeq#:156082205 *Protein ID (NP or XP #) or Wolbachia#: XP_001608591.1 *Organism (including strain): Plasmodium Vivax Etiologic Risk Group (see link below): *Background/Disease Information (sort of like the Intro to your Mini Research Write up): Plasmodium vivax is the most widely distributed cause of malaria in people. There are up to 2.5 billion people at risk and an estimated 80 million to 300 million clinical cases every year. There is another cause to malaria, called Plasmodium falciparum and it is a more extensively researched than Plasmodium vivax even though both contribute to the issue of malaria. However, because it is becoming more recognized now that P vivax is something of concern with the continuous invasion of malaria, there is a new emphasis on studying the parasite. Yet, because of its genetic make up, it is difficult to understand the transmission of P vivax with the lack of information available on its structure and the ways to prevent and treat infections with P vivax. Link to TDR Targets page (if present): http://www.tdrtargets.org/targets/view?gene_id=265333 Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): http://www.ncbi.nlm.nih.gov/nuccore/XM_001608541.1 http://plasmodb.org/plasmo/showRecord.do?name=GeneRecordClasses.GeneRecordClass&primary_key=PVX_093605 Essentiality of this protein: PVX_093605 has essentiality data Gene/Ortholog: Tb927.10.6460 (OG4_10634); Phenotype: no significant loss or gain of fitness in bloodstream forms (3 days); Source study: alsford Gene/Ortholog: Tb927.10.6460 (OG4_10634); Phenotype: significant loss of fitness in bloodstream forms (6 days); Source study: alsford Gene/Ortholog: Tb927.10.6460 (OG4_10634); Phenotype: significant loss of fitness in procyclic forms; Source study: alsford Gene/Ortholog: Tb927.10.6460 (OG4_10634); Phenotype: significant loss of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford Complex of proteins?: No Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Yes, 0.8. By orthology to known druggable targets, there were 4 papers for my target while by sequence similarity to non-orthologous druggable targets, there are 12 papers available. *EC#: 3.1.3.16 Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16&Suchword=&organism[]=Plasmodium+falciparum&show_tm=0 -- Show screenshot of BRENDA enzyme mechanism schematic Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Says to use Enzyme Assay with Calcineurin but according to the experiments already done, a phosphoprotien + H2O = a protein + phosphate reaction should work just as well. This reaction has already been used on the major target of Plasmodium falciparum which is very similar to my target of Plasmodium vivax. Calcineurin uses continuous spectrophotometric rate determination which can be done in our lab but the enzyme assay requires 5 different steps to reach the product of NAD which is too complex, time consuming, and expensive. From Sigma-Aldrich: The cost of 4-nitrophenyl phosphate in disodium salt hexahydrate form for enzyme immunoassay, the price is $80.40. for 5G. The cost of 4-nitrophenyl phosphate bis(tris) salt for the determination of phosphatase/a substrate for the determination of acid and alkaline phosphatase is $108 for 5G. Water would be available in the lab without extra cost.
There is no PDB structure available. However, after doing a BLAST P with the predicted protein sequence of a length of 564 aa found at PlasmoDB (Plasmodium Genomics Resource), there were some matches found but none that were too exact. The best one found was one with Ident: 55%, E value: 2e-134, Query cover: 78%, and called Chain A, Crystal Structure Of The Rat Calcineurin.
Current Inhibitors: Using the TDR Target Page, it was found that:
By orthology to known druggable targets, there were 4 papers for my target while by sequence similarity to non-orthologous druggable targets, there are 12 papers available. Expression Information (has it been expressed in bacterial cells): There is no gene expression information available on the TDR Targets page for my target.
TDR Targets says that the predicted number of trans-membrane segments is 0. However, ExPASy says that the following is the possible transmembrane helices.
The sequence positions in brackets denominate the core region. Only scores above 500 are considered significant.
Inside to outside helices : 6 found from to score center
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MEPLPNPKNDRQVKDVEPPPAKPLSLELLYPNGTDEPPDYKALRDHLKKEGRIRKEDCLD IIKRVIDIVSNEPNLLRLQDPITIVGDIHGQYYDFLKLLEVGGNPDNTQFLFLGDYVDRG SFSIEVLLLLYALKINFPHKIWLIRGNHECRQMTSFFNFRDECEYKYDMVVYYAFMESFD TIPLSAVINGKFLAVHGGLSPQLVLLNQICSFTRFQEPPRSGIFCDILWADPIDEDKEEH TIQTESYFPNDIRGCSYFFGYNAATTFLEKNGLLSIIRAHEAQLEGYKMHQTNLKTGFPI VITIFSAPNYCDVYNNKGAVLKFDSNTLNIQQFSFSPHPYHLPNFMNLFTWSLPFVSEKV TEMLYCILNSSVNQSDEGVKDVVLPAEVLQIISYIEENNVKLEGMTLSGGGGATAGAAST GATEGSPSSQRKEALFKEGCFHSGASKEGGALGTTSPAAATATTQQMAAQGEQPAHLHT DDAQASKERSDALRKKVQSVGRLMRVFRTLRKENELIVQLKGCSPGYRIPVGLLLQGKEG LENELEKFTKAKQIDSINEKRPPNE (564 aa) Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 63258 Da Molar Extinction coefficient of your protein at 280 nm wavelength:
<span style="background-color: #ffffff; color: #626262;">Ext. coefficient 46925
Abs 0.1% (=1 g/l) 0.742, assuming all pairs of Cys residues form cystines
Ext. coefficient 46300
Abs 0.1% (=1 g/l) 0.732, assuming all Cys residues are reduced</span>
*CDS Gene Sequence (paste as text only): "MEPLPNPKNDRQVKDVEPPPAKPLSLELLYPNGTDEPPDYKALRDHL KKEGRIRKEDCLDIIKRVIDIVSNEPNLLRLQDPITIVGDIHGQYYDFLKLL EVGGNPDNTQFLFLGDYVDRGSFSIEVLLLLYALKINFPHKIWLIRGNHE CRQMTSFFNFRDECEYKYDMVVYYAFMESFDTIPLSAVINGKFLAVHG GLSPQLVLLNQICSFTRFQEPPRSGIFCDILWADPIDEDKEEHTIQTESY FPNDIRGCSYFFGYNAATTFLEKNGLLSIIRAHEAQLEGYKMHQTNLKT GFPIVITIFSAPNYCDVYNNKGAVLKFDSNTLNIQQFSFSPHPYHLPNFM NLFTWSLPFVSEKVTEMLYCILNSSVNQSDEGVKDVVLPAEVLQIISYIE ENNVKLEGMTLSGGGGATAGAASTGATEGSPSSQRKEALFKEGCFHS GASKEGGALGTTSPAAATATTQQMAAQGEQPAHLHTDDAQASKERSD ALRKKVQSVGRLMRVFRTLRKENELIVQLKGCSPGYRIPVGLLLQGKE GLENELEKFTKAKQIDSINEKRPPNE" *GC% Content for gene: 8.8652482269504 % *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
*NCBI Gene # or RefSeq#: 156082205
*Protein ID (NP or XP #) or Wolbachia#: XP_001608591.1
*Organism (including strain): Plasmodium Vivax
Etiologic Risk Group (see link below):
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Plasmodium vivax is the most widely distributed cause of malaria in people. There are up to 2.5 billion people at risk and an estimated 80 million to 300 million clinical cases every year. There is another cause to malaria, called Plasmodium falciparum and it is a more extensively researched than Plasmodium vivax even though both contribute to the issue of malaria. However, because it is becoming more recognized now that P vivax is something of concern with the continuous invasion of malaria, there is a new emphasis on studying the parasite. Yet, because of its genetic make up, it is difficult to understand the transmission of P vivax with the lack of information available on its structure and the ways to prevent and treat infections with P vivax.
Link to TDR Targets page (if present):
http://www.tdrtargets.org/targets/view?gene_id=265333
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):
http://www.ncbi.nlm.nih.gov/nuccore/XM_001608541.1
http://plasmodb.org/plasmo/showRecord.do?name=GeneRecordClasses.GeneRecordClass&primary_key=PVX_093605
Essentiality of this protein:
PVX_093605 has essentiality data
Gene/Ortholog: Tb927.10.6460 (OG4_10634); Phenotype: no significant loss or gain of fitness in bloodstream forms (3 days); Source study: alsford
Gene/Ortholog: Tb927.10.6460 (OG4_10634); Phenotype: significant loss of fitness in bloodstream forms (6 days); Source study: alsford
Gene/Ortholog: Tb927.10.6460 (OG4_10634); Phenotype: significant loss of fitness in procyclic forms; Source study: alsford
Gene/Ortholog: Tb927.10.6460 (OG4_10634); Phenotype: significant loss of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford
Complex of proteins?: No
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Yes, 0.8. By orthology to known druggable targets, there were 4 papers for my target while by sequence similarity to non-orthologous druggable targets, there are 12 papers available.
*EC#: 3.1.3.16
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16&Suchword=&organism[]=Plasmodium+falciparum&show_tm=0
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Says to use Enzyme Assay with Calcineurin but according to the experiments already done, a phosphoprotien + H2O = a protein + phosphate reaction should work just as well. This reaction has already been used on the major target of Plasmodium falciparum which is very similar to my target of Plasmodium vivax. Calcineurin uses continuous spectrophotometric rate determination which can be done in our lab but the enzyme assay requires 5 different steps to reach the product of NAD which is too complex, time consuming, and expensive.
From Sigma-Aldrich:
The cost of 4-nitrophenyl phosphate in disodium salt hexahydrate form for enzyme immunoassay, the price is $80.40. for 5G. The cost of 4-nitrophenyl phosphate bis(tris) salt for the determination of phosphatase/a substrate for the determination of acid and alkaline phosphatase is $108 for 5G. Water would be available in the lab without extra cost.
http://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=105298564
Structure Available (PDB or Homology model)
There is no PDB structure available. However, after doing a BLAST P with the predicted protein sequence of a length of 564 aa found at PlasmoDB (Plasmodium Genomics Resource), there were some matches found but none that were too exact. The best one found was one with Ident: 55%, E value: 2e-134, Query cover: 78%, and called Chain A, Crystal Structure Of The Rat Calcineurin.
http://blast.ncbi.nlm.nih.gov/Blast.cgi
http://plasmodb.org/plasmo/showRecord.do?name=GeneRecordClasses.GeneRecordClass&primary_key=PVX_093605
Current Inhibitors:
Using the TDR Target Page, it was found that:
By orthology to known druggable targets, there were 4 papers for my target while by sequence similarity to non-orthologous druggable targets, there are 12 papers available.
Expression Information (has it been expressed in bacterial cells):
There is no gene expression information available on the TDR Targets page for my target.
TDR Targets says that the predicted number of trans-membrane segments is 0. However, ExPASy says that the following is the possible transmembrane helices.
The sequence positions in brackets denominate the core region. Only scores above 500 are considered significant.
Inside to outside helices : 6 found from to score center
120 ( 123) 142 ( 142) 175 132
192 ( 192) 213 ( 210) 648 202
291 ( 295) 311 ( 311) 415 303
340 ( 340) 357 ( 357) 174 348
404 ( 404) 421 ( 421) 558 412
449 ( 449) 467 ( 467) 91 459
Outside to inside helices : 6 found from to score center
116 ( 116) 135 ( 133) 387 125
181 ( 186) 207 ( 204) 470 196
254 ( 259) 277 ( 275) 147 267
296 ( 296) 314 ( 314) 802 304
342 ( 342) 360 ( 358) 1 350
404 ( 404) 422 ( 422) 351 414
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MEPLPNPKNDRQVKDVEPPPAKPLSLELLYPNGTDEPPDYKALRDHLKKEGRIRKEDCLD
IIKRVIDIVSNEPNLLRLQDPITIVGDIHGQYYDFLKLLEVGGNPDNTQFLFLGDYVDRG
SFSIEVLLLLYALKINFPHKIWLIRGNHECRQMTSFFNFRDECEYKYDMVVYYAFMESFD
TIPLSAVINGKFLAVHGGLSPQLVLLNQICSFTRFQEPPRSGIFCDILWADPIDEDKEEH
TIQTESYFPNDIRGCSYFFGYNAATTFLEKNGLLSIIRAHEAQLEGYKMHQTNLKTGFPI
VITIFSAPNYCDVYNNKGAVLKFDSNTLNIQQFSFSPHPYHLPNFMNLFTWSLPFVSEKV
TEMLYCILNSSVNQSDEGVKDVVLPAEVLQIISYIEENNVKLEGMTLSGGGGATAGAAST
GATEGSPSSQRKEALFKEGCFHSGASKEGGALGTTSPAAATATTQQMAAQGEQPAHLHT
DDAQASKERSDALRKKVQSVGRLMRVFRTLRKENELIVQLKGCSPGYRIPVGLLLQGKEG
LENELEKFTKAKQIDSINEKRPPNE (564 aa)
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
63258 Da
Molar Extinction coefficient of your protein at 280 nm wavelength:
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
"MEPLPNPKNDRQVKDVEPPPAKPLSLELLYPNGTDEPPDYKALRDHL
KKEGRIRKEDCLDIIKRVIDIVSNEPNLLRLQDPITIVGDIHGQYYDFLKLL
EVGGNPDNTQFLFLGDYVDRGSFSIEVLLLLYALKINFPHKIWLIRGNHE
CRQMTSFFNFRDECEYKYDMVVYYAFMESFDTIPLSAVINGKFLAVHG
GLSPQLVLLNQICSFTRFQEPPRSGIFCDILWADPIDEDKEEHTIQTESY
FPNDIRGCSYFFGYNAATTFLEKNGLLSIIRAHEAQLEGYKMHQTNLKT
GFPIVITIFSAPNYCDVYNNKGAVLKFDSNTLNIQQFSFSPHPYHLPNFM
NLFTWSLPFVSEKVTEMLYCILNSSVNQSDEGVKDVVLPAEVLQIISYIE
ENNVKLEGMTLSGGGGATAGAASTGATEGSPSSQRKEALFKEGCFHS
GASKEGGALGTTSPAAATATTQQMAAQGEQPAHLHTDDAQASKERSD
ALRKKVQSVGRLMRVFRTLRKENELIVQLKGCSPGYRIPVGLLLQGKE
GLENELEKFTKAKQIDSINEKRPPNE"
*GC% Content for gene: 8.8652482269504 %
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**